An object that actually acts and works in vivo is not a gene, but a protein produced from such gene. Accordingly, clarification and/or analysis of the function and/or structure of a protein are directly associated with, for example, the treatment of disease or drug discovery, and thus are extremely important. Thus, the synthesis and/or production of various types of proteins by various methods, the analyses of the structures of the obtained proteins, and clarification of the mode of action and role of each protein in vivo have vigorously been progressing. At present, it has been well known that the function of a protein is determined not only based on an amino acid sequence constituting it and/or a chain length thereof, but also based on an ordered conformation (higher order structure) obtained from such sequence and/or chain length.
In general, a protein is synthesized using an expression system such as Escherichia coli, insect cells, or mammalian cells. A protein obtained by such synthesis using insect cells or mammalian cells may adopt a controlled higher order structure and an ordered conformation, and may have solubility in many cases. However, such protein synthesis method using insect cells or mammalian cells is disadvantageous in that it is highly expensive and it takes a long period of time to obtain a protein of interest due to extremely complicated separation and purification operations, and also in that the amount of a protein obtained is extremely small. In contrast, in the case of a protein synthesis method using Escherichia coli, operations are simple, it does not take a long period of time to obtain a protein of interest, and it does not cost much. Accordingly, at current, a method using Escherichia coli, into which a genetic code for the synthesis of a protein of interest has been incorporated, is mainly applied to protein synthesis, and the production process thereof is being established.
When a protein of a higher organism such as a human is synthesized using an Escherichia coli expression system, the protein can be obtained as designed, in terms of the binding order of amino acids or the number of amino acids bound, namely, an amino acid chain length. However, the conformation of the obtained protein is disordered, and the higher order structure thereof is not controlled. That is to say, the obtained protein is an insoluble protein known as an “inclusion body,” in which an amino acid chain is entangled. This inclusion body of insoluble protein naturally lacks the desired functions and properties and lacks activity. As a result, an Escherichia coli-based production process requires the refolding of the inclusion body, that is, a process in which the inclusion body is unraveled and converted to a soluble protein with a modulated higher order structure and an ordered conformation.
This type of refolding can be applied not only to a protein produced by Escherichia coli, but to regeneration of a protein that is inactivated by a certain mechanism such as thermal history, and thus it is an extremely important technique. This refolding has been under very active investigation, and different methods have been proposed. Almost all of these methods involve a batch process. Thus, these methods have a low refolding rate and frequently can do nothing more than sporadically give desirable results for certain limited proteins (in particular, for specific low molecular weight proteins). At present, there are no methods for carrying out such refolding, which are universal and general methods applicable to a variety of proteins and which are efficient and economical, providing a high refolding rate.
Under such circumstances, the present inventors have studied a phenomenon whereby a biopolymer selectively adsorbs on an inorganic oxide such as zeolite (see Non-Patent Document 1). During this process, the inventors have found that an inactive protein can be activated by adsorption on and desorption from Zeolite Beta, thereby completing an invention relating to a method for activating the function of an inactive protein using Zeolite Beta (see Patent Documents 1, 2, 3, and 4, and Non-Patent Document 2). However, the methods described in Patent Documents 1, 2, 3, and 4, and Non-Patent Document 2 involve a batch process, and Zeolite Beta is generally obtained in the form of an ultrafine powder particle (a submicron or smaller). Thus, in a separation step in a process in which a protein is allowed to adsorb on or desorb from Zeolite Beta, it is not easy to carry out filtration, and a centrifugal separator must be used many times. The protein activation process of the aforementioned invention has been extremely simple, and it has only utilized adsorption on and desorption from Zeolite. However, this process has been disadvantageous in that the process itself has been complicated and has required time and efforts.
In addition, it has been essential for the conventional refolding operation and/or process to use a centrifugal separator as well as chromatography. Such chromatography has also required time and efforts, and it has resulted in a major problem regarding expensiveness.    [Patent Document 1] JP Patent Publication (Kokai) No. 2005-29531 A    [Patent Document 2] JP Patent Publication (Kokai) No. 2005-192452 A    [Patent Document 3] JP Patent Publication (Kokai) No. 2005-220121 A    [Patent Document 4] JP Patent Publication (Kokai) No. 2005-281267 A    [Non-Patent Document 1] Chem. Eur. J., Vol. 7 (2001) 1555-1560    [Non-Patent Document 2] Anal. Biochem., Vol. 348 (2006) 307-314